This invention relates to methods and apparatus for high throughput sample analysis.
In a range of technology-based industries, including the chemical, bioscience, biomedical, and pharmaceutical industries, it has become increasingly desirable to develop capabilities for rapidly and reliably carrying out chemical and biochemical reactions in large numbers using small quantities of samples and reagents. Carrying out a massive screening program manually, for example, can be exceedingly time consuming and may be entirely impracticable where only a very small quantity of an important sample or component of interest is available, or where a component of a synthesis or analysis is very costly.
Developments in a variety of fields have resulted in an enormous increase in the numbers of targets and compounds that can be subjected to screening.
Rapid and widespread advances in the scientific understanding of critical cellular processes, for example, has led to rationally designed approaches in drug discovery. Molecular genetics and recombinant DNA technologies have made possible the isolation of many genes, and the proteins encoded by some of these show promise as targets for new drugs. Once a target is identified and the gene is cloned, the recombinant protein can be produced in a suitable expression system. Often receptors and enzymes exist in alternative forms, subtypes or isoforms, and using a cloned target focuses the primary screen on the subtype appropriate for the disease. Agonists or antagonists can be identified and their selectivity can then be tested against the other known subtypes. The availability of such cloned genes and corresponding expression systems require screening methods that are specific, sensitive, and capable of automated very high throughput.
Similarly, an emergence of methods for highly parallel chemical synthesis has increased the need for high throughput screening (xe2x80x9cHTSxe2x80x9d). Conventionally, preparation of synthetic analogs to the prototypic lead compound was the established method for drug discovery. Natural products were usually isolated from soil microbes and cultured under a wide variety of conditions. The spectrum of organisms employed by the pharmaceutical industry for isolation of natural products has now expanded from actinomycetes and fungi to include plants, marine organisms, and insects. More recently, the chemistry of creating combinatorial libraries has vastly increased the number of synthetic compounds available for testing. Thousands to tens or hundreds of thousands of small molecules can be rapidly and economically synthesized. See, e.g., U.S. Pat. No. 5,252,743 for a discussion of combinatorial chemistry. Thus, combinatorial libraries complement the large numbers of synthetic compounds available from the more traditional drug discovery programs based, in part, on identifying lead compounds through natural product screening.
Accordingly, considerable resources have been directed to developing methods for high-throughput chemical syntheses, screening, and analyses. A considerable art has emerged, in part from such efforts.
Competitive binding assays, originally developed for immunodiagnostic applications, continue to be commonly employed for quantitatively characterizing receptor-ligand interactions. Despite advances in the development of spectrophotometric- and fluorometric-based bioanalytical assays, radiolabeled ligands are still commonly employed in pharmaceutical HTS applications. Although non-isotopic markers promise to be environmentally cleaner, safer, less expensive, and generally easier to use than radioactive compounds, sensitivity limitations have prevented these new methods from becoming widespread. Another major disadvantage of the competition assay is the number of steps, most notably washing steps, required to run assays.
Scintillation proximity assays, discussed for example in U.S. Pat. No. 4,271,139 and U.S. Pat. No. 4,382,074, were developed as a means of circumventing the wash steps required in the above heterogeneous assays. The homogeneous assay technology, which requires no separation of bound from free ligand, is based on the coating of scintillant beads with an acceptor molecule such as, for example, the target receptor.
In another approach to avoiding the use of radioactive labels, especially useful in high-throughput assays, lanthanide chelates are used in time-resolved fluorometry. See, e.g., U.S. Pat. No. 5,637,509.
Automated laboratory workstations have contributed significantly to advances in pharmaceutical drug discovery and genomic science. See, e.g., U.S. Pat. No. 5,104,621 and U.S. Pat. No. 5,356,525, Particularly, robotics technology has played a major role in providing practical means for carrying out HTS methods. See, e.g., U.S. Pat. No. 4,965,049.
Robotic-based high-throughput tools are now routinely used for screening libraries of compounds for the purpose of identifying lead molecules for their therapeutic potential. For example, a screening method for characterizing ligand binding to a given target employing a variety of separation techniques is described in WO 97/01755, and a related method is described in U.S. Pat. No. 5,585,277.
Highly parallel and automated methods for DNA synthesis and sequencing have also contributed significantly to the success of the human genome project, and a competitive industry has developed. Examples of automated DNA analysis and synthesis include, e.g., U.S. Pat. No. 5,455,008; U.S. Pat. No. 5,589,330; U.S. Pat. No. 5,599,695; U.S. Pat. No. 5,631,734; and U.S. Pat. No. 5,202,231.
Computerized data handling and analysis systems have also emerged with the commercial availability of high-throughput instrumentation for numerous life sciences research and development applications. Commercial software, including database and data management software, has become routine in order to efficiently handle the large amount of data being generated.
With the developments outlined above in molecular and cellular biology, combined with advancements in combinatorial chemistry, there has been a huge increase in the number of targets and compounds available for screening. In addition, many new human genes and their expressed proteins are being identified by the human genome project and will therefore greatly expand the pool of new targets for drug discovery. A great need exists for the development of more efficient ultrahigh throughput methods and instrumentation for pharmaceutical and genomic science screening applications.
Miniaturization of chemical analysis systems, employing semiconductor processing methods, including photolithography and other wafer fabrication techniques borrowed from the microelectronics industry, has attracted increasing attention and has progressed rapidly. The so-called xe2x80x9clab-on-a-chipxe2x80x9d technology enables sample preparation and analysis to be carried out on-board microfluidic-based cassettes. Moving fluids through a network of interconnecting enclosed microchannels of capillary dimensions is possible using electrokinetic transport methods.
Applications of microfluidics technology embodied in the form of analytical devices has many attractive features for pharmaceutical high throughput screening. Advantages of miniaturization include greatly increased throughput and reduced costs, in addition to low consumption of both samples and reagents and system portability. Implementation of these developments in microfluidics and laboratory automation hold great promise for contributing to advancements in life sciences research and development.
Of particular interest are microfluidics devices in which very small volumes of fluids are manipulated in microstructures, including microcavities and microchannels of capillary dimension, at least in part by application of electrical fields to induce I electrokinetic flow of materials within the microstructures. Application of an electric potential between electrodes contacting liquid media contained within a microchannel having cross-sectional dimensions in the range from about 1 xcexcm to upwards of about 1 mm results in movement of the contents within the channel by electroosmotic flow and/or by electrophoresis. Electrophoresis is movement of electrically charged particles, aggregates, molecules or ions in the liquid medium toward or away from the electrodes. Electroosmotic flow is bulk fluid flow, including movement of the liquid medium and of dissolved or suspended materials in the liquid medium. The extent of bulk fluid flow resulting from application of a given electrical field depends among other factors upon the viscosity of the medium and on the electrical charge on the wall of the microchannel. Both electroosmotic flow and electrophoresis can be used to transport substances from one point to another within microchannel device.
Electrophoresis has become an indispensable analytical tool of the biotechnology and other industries, as it is used extensively in a variety of applications, including separation, identification and preparation of pure samples of nucleic acids, proteins, and carbohydrates; identification of a particular analyte in a complex mixture; and the like.
Of increasing interest in the broader field of electrophoresis is capillary electrophoresis (xe2x80x9cCExe2x80x9d), where particular entities or species are moved through a medium in an electrophoretic chamber of capillary dimensions under the influence of an applied electric field. Benefits of CE include rapid run times, high separation efficiency, small sample volumes, etc. Although CE was originally carried out in capillary tubes, of increasing interest is the practice of using microchannels or trenches of capillary dimension on a planar substrate, known as microchannel electrophoresis (xe2x80x9cMCExe2x80x9d). CE and MCE are increasingly finding use in a number of different applications in both basic research and industrial processes, including analytical, biomedical, pharmaceutical, environmental, molecular, biological, food and clinical applications.
Typically, the microchannels of MCE devices are constructed by forming troughs or grooves of appropriate dimension and configuration in one surface of a planar rectangular- or disc-shaped base substrate, and applying a planar cover to the surface to enclose the microchannels.
Conventionally, microchannels having capillary dimensions have been made in silicon or glass substrates by micromachining, or by employing photolithographic techniques. See, e.g., U.S. Pat. Nos. 4,908,112, 5,250,263. Where the substrates are of fused silica, the microchannels can be enclosed by anodic bonding of a base and a cover. Exemplary MCE devices are also described in U.S. Pat. Nos. 5,126,022; 5,296,114; 5,180,480; and 5,132,012; and in Harrison et al., xe2x80x9cMicromachining a Miniaturized Capillary Electrophoresis-Based Chemical Analysis System on a Chip,xe2x80x9d Science (1992) 261: 895; Jacobsen et al, xe2x80x9cPrecolumn Reactions with Electrophoretic Analysis Integrated on a Microchip,xe2x80x9d Anal. Chem. (1994) 66: 2949; Effenhauser et al., xe2x80x9cHigh-Speed Separation of Antisense Oligonucleotides on a Micromachined Capillary Electrophoresis Device,xe2x80x9d Anal. Chem. (1994) 66:2949; and Woolley and Mathies, xe2x80x9cUltra-High-Speed DNA Fragment Separations Using Capillary Array Electrophoresis Chips,xe2x80x9d P.N.A.S. USA (1994) 91:11348,
Eckstrom et. al. U.S. Pat. No. 5,376,252 describes a process for creating capillary size channels in plastic using elastomeric spacing layers. Ohhman International Patent Publication WO 94/29400 describes a method for producing microchannel structures by applying a thin layer of a thermoplastic material to one or both of the surfaces to bejoined, then joining the surfaces and heating the joined parts to melt the thermoplastic bonding layer. Kaltenbach et al. U.S. Pat. No. 5,500,071 describes constructing a miniaturized planar microcolurn liquid phase analytical device by laser ablating microstructures in the surface of a planar laser ablatable polymeric or ceramic substrate, rather than by conventional silicon micromachining or etching techniques.
U.S. Pat. No. 6,176,962 describes methods for fabricating Mnicrochannel structures constructed of a polymeric card-shaped or disc-shaped base plate having a planar surface in which a microchannel structure is formed, and a planar polymeric cover. The microchannel structure is enclosed by bonding the planar surfaces of the cover and the base plate together.
In one general aspect, the invention features a continuous form microstructure (i.e., microcavity and/or microchannel) array device constructed as an elongate flexible film laminate containing a plurality of microstructures or arrays of microstructures arranged serially lengthwise along the laminate. Where the device has a series of microstmctures, each structure is configured to carry out a fluidic process or a step in a fluidic process. Where the device has a series of microchannel arrays, each array is configured to carry out a set of processes or steps, on an array of samples or of test reagents.
Because the microstructures, or arrays of microstructures, are serially arranged lengthwise along the laminate, the device can be fed lengthwise into and through an analytical device, and the structures or arrays can be treated serially in a continuous automated or semiautomated manner.
In some embodiments the flexible elongate laminate device is advanced within the analytic device from a continuous uncut supply roll, through the various parts of the analytical device and, as the laminate device is expended, to a takeup roll, similar to the way in which roll film is advanced frame-by-flame through a camera In other embodiments the elongate laminate device is advanced within the analytic device from a continuous uncut accordion-folded supply stack, through the analytical device and, as the laminate device is expended, to an accordion-folded takeup stack. When the entire roll (or supply stack) has been expended and passed onto the takeup roll (or stack), the expended roll (or stack) can be discarded, or can conveniently and efficiently be stored in an archive, as may be desirable for some uses.
The microstructures are constructed either by forming channels, trenches or cavities of suitable dimension and configuration in a microchannel surface of a first lamina and, optionally, enclosing the channels by apposing a covering surface of a second lamina onto the microchannel surface to form the microstructures; or by forming slits having suitable dimension and. configuration in a spacing lamina, and sandwiching the spacing lamina between first and second enclosing laminae to enclose the slits between the apposed surfaces of the first and second enclosing laminae to form the microchannels.
Electrodes can be formed in the device by any of a variety of techniques, known in the art, including application of wires or conductive decals, or printing or stamping using conductive inks, or vapor deposition, etc., in a specific configuration onto a surface of one or both of the laminae. The laminate construction according to the invention is particularly suitable for application of flexible printed circuit technology. For technical review, See, Th. H. Stearns (1996), Flexible Printed Circuitry, SMTnet Bookstore. See also, U.S. Pat. Nos. 4,626,462; 4,675,786; 4,715,928; 4,812,213; 5,219,640; 5,615,088.
Processes for making flexible printed circuits are generally well known. Briefly, the electrodes, which provide connections from the reservoirs in the microfluidic structure to high-voltage contacts in an analytical device that carried the laminate, are formed within a thin polymer film laminate, which serves as a cover lamina to be affixed as described above to the base lamina, as described in more detail below.
In this context, an xe2x80x9canalytical devicexe2x80x9d is a device that includes at least a detector capable of detecting or of measuring a signal produced in the course of the microfluidic process or process step, and means for moving the laminate in relation to the analytical device to bring a detection region in the microstructure within the field of the detector. Usually the analytical device is in a stable installation, and the laminate is advanced through it past the detector, but in some embodiments the laminate is held in place and the analytical device is moved along it. Of course, any number of such detectors may be employed, each alignable with a detection region (or series of detection regions, as the laminate progresses through). Usually, the analytical device also includes electrical contacts each alignable with a contact point in electrical circuitry employed to generate electroflow in the microstructure. Each such contact is electrically connected to a source of electrical power, and to control means (which may be automated) for changing the applied electric fields as the microfluidic process proceeds. The analytical device may further include means for adding various fluids (e.g., samples, buffers or other solvents, reagents, and the like) to the microstructures by way of access ports in the laminate. The analytical device may additionally include means for changing the environmental conditions surrounding a portion of the laminate, such as temperature, and the like.
In some embodiments, the device is provided as an assembled laminate, in which the microchannels are fully enclosed; and in which ports or reservoirs are provided for introduction of sample or reagents or test compounds or liquid media; and in which electrodes have been emplaced and provided with leads for connection to a source of electrical power. Reagents, samples, test compounds, and/or media can be introduced as appropriate during or just prior to conducting the assays. In some embodiments the assembled laminate is provided with at least some of the media or reagents xe2x80x9con boardxe2x80x9d in the microchannels or reservoirs as appropriate. Where the device is provided with one or more substances already on board, the device can additionally be provided with means for protection of degradable contents from variations in ambient conditions and, particularly, for example, a release liner which resists loss of moisture or of volatile contents and/or which resists light exposure to the contents, may be provided as a release liner on one or both surfaces of the laminate.
The device and method of the invention provides a full range of advantages in analytical sensitivity that inhere in the use of conventional microfluidic analysis, while at the same time providing for automated or semiautomated continuous processing of high numbers of analyses at high rates of speed. The complexity of mass screening programs, for example, is substantially reduced by elimination of many of the manipulation steps, whether by hand or by machine, that are required in use of conventional assay plates. And possibilities for error are reduced by reduction of the number of points at which manipulation by hand is required.
Methods and apparatus according to the invention for carrying out multiple microfluidic manipulations at high throughput rates are readily adaptable for automated non-contact dispensing of reagents or samples, providing for substantially reduced risk of cross-contamination.
Further, the continuous form assay array according to the invention significantly reduces the bulk volume of disposable materials, as compared with conventional assay card methods, both because the flexible laminates themselves are thinner than are conventional assay cards, and because the microchannel structures or arrays can be arranged on the continuous form device with more efficient use of the substrate surface area.